Nitric Oxide Signaling Mediates Stimulation of L-Type Ca(2+) Current Elicited by Withdrawal of Acetylcholine in Cat Atrial Myocytes

A perforated-patch whole-cell recording method was used to determine whether nitric oxide signaling participates in acetylcholine (ACh)-induced regulation of basal L-type Ca(2+) current (I(Ca,L)) in cat atrial myocytes. Exposure to 1 μM ACh for 2 min inhibited basal I(Ca,L) (−21 ± 3%), and withdrawal of ACh elicited rebound stimulation of I(Ca,L) above control (80 ± 13%) (n = 23). Stimulation of I(Ca,L) elicited by withdrawal of ACh (but not ACh-induced inhibition of I(Ca,L)) was blocked by either 50 μM hemoglobin; 30 μM ODQ or 10 μM methylene blue, inhibitors of soluble guanylate cyclase; 10 μM W-7, a calmodulin inhibitor; or 10 μM L-NIO, an inhibitor of constitutive NO synthase (NOS). In cells incubated in 5 mM l-arginine, ACh-induced rebound stimulation of I(Ca,L) was enhanced compared with control responses. Histochemical assay (NADPH diaphorase) indicated that atrial myocytes express constitutive NOS. NO-donor, spermine/NO (SP/NO), >1 μM stimulated basal I(Ca,L). SP/NO-induced stimulation of I(Ca,L) was inhibited by 50 μM hemoglobin, 30 μM ODQ, or 5 μM H-89, an inhibitor of PKA, and was unchanged by 50 μM MnTBAP, a peroxynitrite scavenger. When I(Ca,L) was prestimulated by 10 μM milrinone, an inhibitor of cGMP-inhibited phosphodiesterase (type III) activity, SP/NO failed to further increase I(Ca,L). In cells incubated in pertussis toxin (3.4 μg/ml for 6 h; 36°C), ACh failed to affect I(Ca,L), but 100 μM SP/NO or 10 μM milrinone still increased basal I(Ca,L). These results indicate that in cat atrial myocytes NO signaling mediates stimulation of I(Ca,L) elicited by withdrawal of ACh but not ACh-induced inhibition of basal I(Ca,L). NO activates cGMP-induced inhibition of phosphodiesterase (type III) activity. Upon withdrawal of ACh, this mechanism allows cAMP to recover to levels above control, thereby stimulating I(Ca,L). Pertussis toxin–sensitive G-proteins couple M(2) muscarinic receptors to NO signaling. NO-mediated stimulation of I(Ca,L) elicited by withdrawal of ACh may be an important mechanism that rapidly restores cardiac pacemaker and contractile functions after cholinergic suppression of atrial activity.